Supplementary Table 2: ALK mutations in neuroblastoma cell lines and comparison

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1 Supplementary data Supplementary Table 2: ALK mutations in neuroblastoma cell lines and comparison with published studies. (MNA = MYCN amplification, A = MYCN amplified, NA = not MYCN amplified, wt = wild-type). The last column describes the source from where the cell lines were obtained : 8 cell lines from the lab of Peter White (The Children s Hospital of Philadelphia, Philadelphia, PA), 1 cell line from the lab of Valérie Combaret (Department of Cellular Biology, Lyon, France), 17 cell lines from the lab of ogier Versteeg (Department of Human Genetics, Amsterdam, The Netherlands), 3 from the lab of Susan agsdale (St. Jude Children s esearch Hospital, Memphis, TN), 1 from the lab of Susan L. Cohn (Department of Pediatrics, Chicago, IL), 1 from the lab of Sven Påhlman (Center for Molecular Pathology,Malmö, Sweden), 1 from the lab of John Lunec (Northern Institute for Cancer esearch, Newcastle upon Tyne, UK), 5 from the lab of Peter Ambros (Cancer esearch Institute, Vienna, Austria), 1 from the lab of Frank Speleman (Center for Medical Genetics, Ghent, Belgium), 1 from Panarello (Istituto Giannina Gaslini, Genova, Italy), 1 from the Japan Health Sciences Foundation (JHSF) and 1 from the American Type Culture Collection (ATCC). All cell lines tested negative for Mycoplasm in September 2008.

2 ID MYCN status ALK amplification? ALK mutation? Mutation confirmed in other studies? Source of cell lines CHP-134 A no wt White CHP901 A no wt White CHP902 A no wt White CLB-GA NA no 1275Q Combaret GICIN gain no wt Panarello Gl-ME-N NA no wt Versteeg IM32 A partial amplification wt Versteeg LAN-1 A no F1174L Versteeg LAN-2 A no wt Versteeg LAN-5 A no 1275Q 8 Versteeg LAN-6 NA no D1091N 7 White N206 A no F1174L 10 Versteeg NB-1 NA amplification wt JHSF NB-5 A no wt agsdale NB-13 A no wt agsdale NB-14 A no F1174L agsdale NBL-S NA no wt White NGP A no wt Versteeg NLF A no wt White NMB A no wt White SK-N- SH/SHEP/SH- SY5Y NA no F1174L 10 7,8,10 7,8,10 Versteeg/ Cohn/ Pålman SJNB-1 NA no wt Versteeg SJNB-6 A no wt Versteeg SJNB-8 A no wt Versteeg SJNB-10 A no wt Versteeg SJNB-12 NA no wt Versteeg SK-N-AS NA no wt ATCC SK-N-BE A no wt Versteeg SK-N-BE(2c) A no wt Lunec SK-N-FI NA no wt Versteeg SMS-KAN A no wt White SMS-KCN A no F1174L 10 / George et al. (1275Q) Versteeg STA-NB-3 A no wt Ambros STA-NB-8 A no F1174L Ambros STA-NB-9 A no wt Ambros STA-NB-10 A no wt Ambros STA-NB-12 NA no wt Ambros T14 A no wt Versteeg UHG-NP A no wt Speleman

3 Supplementary Table 3: Primers used for amplification and sequencing of the ALK gene. Exon Forward or everse Primer sequence 5 3 Exon 1 F CGCGGCTCGCTAGCTC GAACTGCGTTCAGGGAGAAAAC Exon 2 F GGGTCCTGAGGTCAACTCAGTC CATATAGGGAGCTGAGGGAATGC Exon 3 F AAGGCCAACCTCCTAGTGTGG AGAGCAGAACAGGAGTGAAGTCTCAT Exon 4 F GCAGAGAAGGAGATAATGGCCTC GGACCTACCGATTAAAGGACAATC Exon 5 F CCTGAAGTCCCTGCAAGGA AAACATGGTTGCAGGTTATTGAC Exon 6 F TCAGGCCTTGGAGTGTGAATG GCTTTGCCATGAGAGGAGTCA Exon 7 F CAGTGTCATTAGTCATCCAGCATTC AAGCTTTGGTCAGACACCCG Exon 8 F TTGACAGGTGGGCTTTCTTCTC CCAGCCAGCTAGGCTACTTTCT Exon 9 F TCCAGCTCTTCTCACCAGGC CTGCATGTGTGTCTTGGGTAAAA Exon F GGTGATCAGCACACACCTGC AGCACCAATCTTTCTTCTGCCT Exon 12 F TCGATGGCTGATTGGTGTTTC CTATGGGCCTGAGTCTGTTGC Exon 13 F TAAACTTGATCGTCCTTTCCTGAA TTGCTGTCTCATTCTCCTGGTATG Exon 14 F ATTTTTCTGCCTCCTCAACTCAA CTGTACCTGGCCCTGCTCAT Exon 15 F GTAACAAGGGTTCAGAGCCCG AGTGACTAAGATGCCCTCAGGC Exon 16 F TTTCCTAGTGTGGATCTGAAGCC TCACAGTCCACACTTGGGCA Exon 17 F CCCCAGTGACCCCCTAACTT CTGCGCCATAGGAAGCTTG Exon 18 F ACAGTTGTGCTTCCAGTGGCT TTTACAAAACCGAATCCAGGGT Exon 19 F CTGTCAAATCGGGATGAGTCTG AATTCCAGGGACTAGCATAACGAA Exon 20 F GGAAAGGTTCAGAGCTCAGGG GGCCTTTTGTGGCTAGAGGAGT Exon F GTGTGTATGTTACCCCCGCCT ACATGCTAGGGACAACACGATTT Exon 23 F GGAGCCTGCTGTGGTTCTTC AGTTGACACCCTGGGTTCC Exon 24 F CCCCTGGTGTAGCTGCATGT GAAATGTGAGCCCTTGAGATCTG Exon 25 F GGAAATATAGGGAAGGGAAGGAACTA TGATGTAAGGGACAAGCAGCC Exon 26 F ATTTCAGACCTTTAATGGGTAGACTATATTGT CCCGGCTTAGAGTATAGAGTCCTTT

4 Exon 27 F TTTAAGAGTTCTATGTTATGGAAAAGGGTA AAGAAGCATATGTGGCTCTGGATA Exon 28 F TCCTTGACCAATCAGCAGGG CTTGTACTCTGACTGGCTTGACCTAT Exon 29 F TGACTTCCCATTCATTTTGTATGC GTGGGCTTGTTTCTGGATCC

5 Supplementary Figure 1: ALK mutation and amplification frequencies and type in the different published studies and this study (wt = wild type, amp = amplification).

6 P < P < % 18% 16% 14% 12% 10% 8% 6% 4% 2% wt amp 1275 F1174 Y1278 F1245 T % Supplementary Figure 2: Frequencies and types of ALK mutations according to MYCN status and genomic subgroups (wt = wild-type ALK, amp = ALK amplification, MNA = MYCN amplification).

7 Supplementary Figure 3: Alignment of 2p gained regions present in 49 out of 254 neuroblastoma tumors, detected using arraycgh. Co-occurrence with MYCN amplification is marked with a star.

8 Supplementary Figure 4: Comparison of Affymetrix exon array ALK expression with arraycgh copy number data at the ALK position of 101 neuroblastoma tumors (X- and Y-axis = log scale, DOD = dead of disease).

9 Supplementary Figure 5: Correlation plots of ALK mna levels (A) or ALK protein levels (B) versus phospho-alk (palk) status. The black dot represents the NB1 cell line which has an ALK amplification, red dots represent F1174L mutated cell lines, green dots 1275Q mutated cell lines and blue dots ALK wild-type cell lines. Only wild-type cell lines with a clear ALK protein expression on Western blot were included.

10 Supplementary Figure 6: Western blot analysis of ALK (A) and phospho-alk (palk) (B) status in 22 neuroblastoma cell lines. 7 cell lines are ALK mutated (underlined), 1 has an ALK amplification (NB1) and 14 are ALK wild-type. Anti-EK2 was used as loading control (C). ALK blots were exposed for 5 minutes and palk blots for 20 minutes. ( cell lines with F1174L mutation, _ cell lines with 1275Q mutation).

11 amp F wt Supplementary Figure 7: Box-plots representing relative phospho-alk levels (palk/alk) in NB cell lines with different mutation status. (amp = amplification, wt = wild-type)